The use of solid supports in chemistry and affinity chromatography has received much attention in recent years. These methodologies involve the derivatization of a solid matrix and usually a subsequent chemical modification of the introduced functionalities. Since these solid matrices are generally insoluble in most solvents, evaluation of those transformations is problematic. Common questions in this regard involve quantitative concerns about titers of ligands and their long term stability on the support or qualitative questions regarding structural features of the ligands.
Some methods for evaluating transformed supports have been reviewed (Practical Guide For Use in Affinity Chromatography and Related TechniquesIBF/LKB pp. 43-45 (1983) and prominently include acid-base and redox titrations, microanalysis and indirect evaluation by difference. These methods are severely limited when the support is derivatized to a very small extent and generally do not afford structural information regarding the relevant ligand. This is so because the usual spectroscopic methods such as NMR do not work. Specific applications such as solid phase peptide and oligonucleotide synthesis are evaluated by cleavage of support-bound products followed by assays available to regular practitioners of these technologies. However, a generally applicable assay is not available since the chemistry involved in such an assay would have to be compatible with the large diversity of ligands and supports.
Cleavable bifunctional molecules are well known for their use in mapping contact sites of biomacromolecules (Pierce Handbook pp. 221-250, (1988) Pierce, Rockford, IL) and they have also been reported useful in affinity chromatography when elution of very tightly bound ligands is required (Jayabaskaran et al., Preparative Biochem., 17, pp. 121-141 (1987); Mouton et al., Biophys., 218, pp. 101-108 (1982); Singh et al., Biophys., 218, pp. 284-293 (1979); Herman et al., Anal. Biochem., 156, pp. 48-55 (1986), for purification of macromolecular reagents (Schwarzberg, U.S. Pat. No. 4,272,506 (1981), for reversible formation of synthetic vesicles (Chang et al., Chem. Letters pp. 1385-1388 (1987), and for reversible immobilization of enzymes (Carlson et al., Hind.Antibiotic Bull. 20, pp. 105-108 (1978)). The most common bond that is split in these linking) molecules is a disulfide moiety.
It is a purpose of the present invention to provide a solid support chemistry system which can be used to define titers of ligands using cleavable, bifunctional molecules.
It is also a purpose of the present invention to provide a solid support chemistry system which can be used to define chemistry distal to the support using cleavable, bifunctional molecules.